Generation of mesenchymal stem cells as a medicinal product in organ transplantation

Abstract

Purpose of review 

Mesenchymal stem cells (MSCs) are emerging as an alternative treatment in solid-organ transplantation. The use of MSCs as a therapeutic product requires the translation of basic research protocols into a production process under good manufacturing practice (GMP) to obtain a safe product of high quality. This requires a different mindset from the academic setting of changing protocols into a well defined, controlled and documented process. This review describes some of the challenges faced by culturing MSCs as a medicinal product.

Recent findings 

Clinical-grade MSCs are used in the clinical trials and proved to be safe as a medicinal product. Because of the differences in the type of MSCs and in the production process, clinical outcome is not always comparable. New standardized methods in the culture condition such as the use of alternatives for fetal bovine serum (FBS), standardized plating densities or the use of bioreactors may further standardize the production process.

Summary 

To generate MSCs as a medicinal product in organ transplantation, regulation requires that MSCs have to be generated under GMP. During the whole production process, all critical steps should be known and described. Further steps should be taken to optimize and standardize the production process.

INTRODUCTION

Mesenchymal stem cells (MSCs) are multipotent stromal cells and are relatively easy to obtain from a variety of tissues. They expand rapidly in culture for a longer period of time, are immunologically inert and have immune-modulating and regenerative potential ^[1–3]. These characteristics render them attractive candidates for cell therapy in organ transplantation in reducing ischemia–reperfusion injury, acute or chronic rejection and in doing so, eventually resulting in long-term graft function ^[4–7].

Culturing human MSCs in a standard conventional laboratory is relatively easy. Translating research-based protocols into the required protocols for obtaining clinical-grade MSCs is something most researchers underestimate. The tissue of which the MSCs will be isolated, materials used for culturing, equipment, culture procedure, validation and not to mention the cost involved are all aspects one has to consider. As the number of clinical trials in which MSCs are used quickly rises, with more than 250 clinical trials shown in the public database www.clinicaltrials.gov of which at least 6 studies involve organ transplantation, the need for a consensus regarding the production of MSCs is needed. Small variations in the manufacturing procedure may result in inconsistent clinical studies and may hamper the regular use of MSCs in clinical applications. Cell therapy is relatively new in healthcare and the essential infrastructure, that is, scalable manufacturing, appropriate regulation, reimbursement and clinical acceptance, is still in development ^[8]. Specifically for MSCs, there are many variations in isolation and culturing MSCs, and an abundance of therapeutic potential of MSCs makes that there are still quite some issues to be resolved. Some of the issues, for the production of MSCs as medicinal products, will be touched upon in this review.

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MESENCHYMAL STEM CELLS AS A BIOLOGICAL MEDICINAL PRODUCT: REGULATION

When MSCs are used to treat or prevent diseases in human beings or when these cells are used to exert a pharmacological, immunological or a metabolic action, as would most likely be the case in organ transplantation, they are considered a biological medicinal product. When MSCs are substantially manipulated or are used for another essential function in the recipient than in the donor, they are regarded in Europe as an Advanced Therapy Medicinal Product (ATMP) ^[9]. The use of ATMPs is subjected to regulation 1394/2007, amending directive 2001/83/EC (Annex 1 replaced by 2009/120/EG) and regulation 726/204. These regulations include rules regarding the safety of a product, validation, efficacy and traceability and production that has to be in compliance with good manufacturing practice (GMP) according to the directive 2003/94/EC (www.europa.eu).

There are some differences between the rules in de EU and the USA. In the USA, MSCs are considered human cells, tissues, or cellular and tissue-based products (HCT/Ps) as specified in the definitions of The Code of Federal Regulation (CFR) Title 21 §1271 (21 CFR 1271). When HCT/Ps do not meet the criteria as outlined in 21 CFR 1271.10, they are subjected to licensure or, when in a developmental stage, subjected to the investigational new drug (IND) requirements as regulated under 21 CFR 312 (www.accessdata.fda.gov). This will mostly be the case in using MSCs in organ transplantation as MSCs in these treatments are intended for nonhomologous use. Hence, the production of MSCs must comply with current good tissue practice (cGTP).

Both GMP and cGTP refer to a production process of MSCs that needs to be controlled and documented in detail and performed by qualified persons in a controlled environment.

These regulations are intended to ensure a safe and pure product of high quality, a product that has a consistent efficacy, and a reproducible and robust production process.

CLINICAL-GRADE MESENCHYMAL STEM CELLS

The use of MSCs on a clinical basis mostly demands scaling up the amounts of cells needed by culturing the MSCs. Bone marrow aspirate contains around 6 × 10⁶ cell/ml of which 0.01–0.001% are MSCs and even the richest source of MSCs, the adipose tissue, contains around 0.05–2 × 10⁵ MSCs per gram tissue. These amounts are not sufficient to reach the required dose to treat patients. This dose varies mostly around 1–10 × 10⁶ cells/kg bodyweight, a concentration range that is well tolerated and effective in graft-versus-host disease (GvHD) and Crohn's disease ^[10,11]. Culturing cells means a substantial manipulation and requires a production of MSCs under GMP and, as mentioned, a robust production process. Before staring the culture, one has to consider the product that has to be prepared and the clinical application strived for. First, what is the ‘active ingredient’? Cultured MSCs still lack specific markers. The International Society of Cellular Therapies (ISCT) defined MSCs by their capacity to adhere to plastic, expression of specific phenotypic markers such as CD73, CD105, CD90 (≥95%) and lack of expression of CD14/CD11b, CD34, CD45, CD79α/CD19, HLA-DR (≤2%) and their multipotential differentiation potential ^[12]. Keeping this in mind, let us explore the different sources of MSCs. MSCs could be derived from a number of tissues. Primarily, they are obtained from bone marrow and adipose tissue, but they could also be isolated from umbilical cord blood, placenta, synovial membrane, and numerous other tissues ^[13–16]. These MSCs are all effective in having profound immune-modulating effects ^[14,15,17] and meet the criteria as defined by the ISCT. However, they may have differential functional properties ^[17–19]. Isolated MSCs are in fact a heterogeneous cell population and composition varies with the source of the cells, the isolation and the culturing method. Substantial changes in culture condition may not only favor other (adherent) cell populations such as multipotent adult progenitor cells (MAPCs), but also could skew the cell population into a different cell type ^[20]. Potentially, any manipulations may affect the MSCs and their potential working mechanism ^[21–24]. Attention must be given to the composition of the cell population at critical stages, during and at the end of the culture. Nonadhered cells are lost in the course of media change but will be present in the first culturing period and influence the first passages. In contrast, unwanted adherent cells, mainly macrophages and endothelial, expand together with the MSCs. M1 and M2 polarized macrophages have profound effect on the proliferation being either negative (M1) or positive (M2) ^[25,26]. In addition, quickly dividing cells are rapidly lost early in the culture ^[27▪▪]. The question remains what one wish to obtain in the end. A heterogeneous population may just be needed for a therapeutic effect as early passages could have a better clinical effect than latter passages ^[28▪]. However, a heterogenic cell population that varies in its composition makes a reproducible and robust culturing procedure a greater challenge. Before starting the production of MSCs on a larger scale, one must first define the source of the MSCs and the expected clinical outcome parameters and determine of these fit the production process.

PRODUCTION OF MESENCHYMAL STEM CELLS UNDER GOOD MANUFACTURING PRACTICE

Production under GMP ensures that the product is safe and pure, and the production process reproducible and robust. During the entire process, products and material should be protected from contamination and in-process controls should demonstrate product and production consistency. The production process could be separated into different stages. First, the MSCs have to be isolated from the tissue of choice: bone marrow (BM)-MSCs, adipose tissue (AT)-MSCs or other tissue-MSCs, all require different isolation steps to separate the MSCs from the connective tissue or other cell types. For example, BM-MSCs require a density-gradient step, whereas AT-MSCs are separated by digestion with collagenase. This is in all cases followed by the initial plating, harnessing the adherence properties of MSCs onto plastic. Next, there is the expansion stage of the MSCs. In this stage, the culturing conditions are crucial for maintaining the functional properties of the MSCs ^[24]. Critical factors in the expansion stage are the initial cell density, doubling rate, cell confluence, culture duration and use of FBS, human serum or other substitutes which could potentially influence the cell characteristics and cell doubling time and proliferation rate. Finally, the cultured MSCs are harvested. When MSCs are used for future treatments, they may undergo a preservation step. In all stages, the manufacturing requires the use of products throughout the process which are well defined and documented and of GMP grade. During the whole process, all critical steps should be known and described. As a guideline, some are described below.

Plating density

The initial cell-plating density and the subsequent seeding density of the passages may affect the proliferation rate and the purity of the MSCs ^[29]. For clinical scale production, one wants to obtain high numbers of cells within a short time frame to reduce potential risks such as transformation or contamination of the cells and the substantial cost involved in using a GMP facility for longer periods of time. Adipose tissue has the benefit of containing large amount of MSCs, thereby reducing the need for multiple passages and subsequently less chance of inducing abnormalities. On the other hand, high cell densities may reduce the proliferation rate and purity by loss of multipotency ^[30–32]. Most frequently used cell densities are 160 000 cells/cm² initially and 4000 cells/cm² during each pass ^[10,11].

Culture media

MSCs are typically cultured in Dulbecco's Modified Eagle Medium or alpha-MEM with the addition of FBS as a source of growth factors, cytokines and mitogens ^[33]. For clinical applications, however, the use of FBS harbors some difficulties. FBS batches differ from time to time which could profoundly impact the proliferation rate and hamper reproducibility and consistency of the production process. Another issue is the risk of contamination of FBS with mycoplasma, viruses and specifically bovine spongiform encephalopathy. In addition, FBS has the risk of transferring xenogenic proteins into the patient and inducing host immune responses against foreign antigens. Therefore, regulations require an alternative source when possible. These risks could be avoided by using human alternatives. These are offered in the form of human-blood-derived products such as human platelet lysates ^[34,35], human plasma and serum or cord blood serum ^[36▪]. New GMP compliant commercially available serum-free and xeno-free media are available that support or even enhance the proliferation of the MSCs while maintaining the MSCs characterizations ^[37]. These products with names as ‘StemPro MSC’, ‘MesenCult-XF’ and ‘CellGro MSC’ are well defined and, when proven to be efficient in maintaining the culture and the therapeutic properties of MSCs, could markedly reduce the variations in the cell culture and potentially the clinical outcome ^[38]. However, most clinical studies performed with MSCs so far have been cultured with FBS. All alternatives should be tested thoroughly before entering a clinical trial.

Genetic (in)stability

In general, successive passages or long-term cultures could induce genetic instabilities and transformation of cells. Concerns rise that this may also be true for MSCs and that the extensive in-vitro expansion of MSCs may induce malignant formation. Recent studies show that aneuploidy occurs in in-vitro cultured MSCs without any features of transformation, independent of the culture condition ^[39,40]. In addition, MSCs undergoing cell doublings up to 20–25 times or MSCs that become senescent do not show any transformation in vitro or in in-vivo toxicity studies in animals ^[39,41,42]. Eventually, all cultured MSCs become senescent and to avoid any risks, harvest of the clinical grade MSCs should be done before. These data indicate that the transformation of MSCs is uncommon in-vitro culture. Still, for safety reasons, the genetic characteristics of MSCs should be tested before the release of the MSCs for clinical use in standard karyotyping, fluorescence in situ hybridization or single-nucleotide polymorphism analyses ^{[40,43▪▪,44]}.

Product release

Before release of a drug, clearly defined release specification are needed and hence a defined set of release tests. During the whole process, the MSCs should be monitored for possible contamination and especially upon the final release of the clinical-grade cells, they must be free of bacterial, endotoxin, viral, fungi and mycoplasma contamination and free of impurities such as other unwanted cell types. Other easy to obtain criteria are viability, genetic stability and, as they need to be identified as MSCs, phenotypical characterization by flow cytometric analysis. More time-consuming criteria are osteogenic, chondrogenic or adipogenic differentiation capacities and testing for clonogenicity of the obtained cells. When cells are used freshly, the release criteria have to be defined within a reasonable timeframe and because of this time constraint, these assays may be less practical. Furthermore, the cells must exert their presumed pharmacological effect related to the intended clinical use. However, in-vitro assays do not always correlate with the presumed clinical effect as is observed, for example, for the immunosuppressive capacities and these criteria may be less useful ^[28▪,42]. Eventually, potency assays will mostly compromise a combination of functional assays and phenotypic assays.

STANDARDIZATION OF THE CULTURE PROCEDURE

It has often been suggested to develop an international consensus for culturing MSCs. No doubt that the international guidelines are needed to be able to compare the outcome of clinical trials. But standardized protocols need to be directed on the intended clinical use in combination with the source of the MSCs. In a large multicenter trial for the treatment of GvHD, different production sides use the ex-vivo expansion procedure as defined by the European Group for Blood and Marrow Transplantation ^[10]. The same should be developed for the use of MSCs in organ transplantation.

‘OFF-THE-SHELF’ MESENCHYMAL STEM CELLS

Using ‘off-the-shelf’ MSCs is also a possibility, bypassing the need for the difficult cost and time-consuming production process of personalized MSCs. The MSCs are derived from the healthy donors and expanded extensively. Various companies such as Athersys, Genzyme or Osiris have their products in clinical trials or even approved (Prochymal) as stem cell drug for GvHD. Efforts are undertaken to develop an off-the-shelf MSCs for use in solid-organ transplantation ^[45].

DEVICES FOR PRODUCTION

Culturing MSCs is often performed in culture flasks or an ‘open system’. This procedure is labor intensive and always has the risk of contamination. Hence, the procedures are confined to strict environmental conditions. Closed, automatic devices or bioreactors are of great interest to produce large amounts of cells in a short timeframe and to reduce the workload and cost of the production process. Closed bioreactors will minimize the handling of the propagation and doing so, reduce the risk of unwanted contaminations. Different methods are developed varying from stirred suspension using microcarriers, roller bottles, three-dimensional microcarriers or hollow fibers ^[46–48]. Optimizations of the culture condition of MSCs are based on culturing the cells in a monolayer in which expansion is limited by the surface array. In bioreactors, the cells are cultured in a markedly different way. The oxygen exchange, sheer stress caused by hydrodynamics or pH are critical factors in the bioreactors and different from the monolayer method. MSCs cultured in bioreactors show a different morphology and changed differentiation capacity ^[48]. Several issues need to be addressed before MSCs cultured in bioreactors could be used for the clinical applications. Nevertheless, bioreactors may be the success needed for implementing personalized medicine in the clinic that could compete with off-the-shelf MSCs.

CONCLUSION

Since the first report from Friedenstein of clonogenic potential of multipotent marrow, MSCs are used for an abundancy of therapeutic indications ^[49]. Many clinical trials have been performed without major adverse effects showing MSCs treatment is well tolerated. Still, some obstacles have to be taken regarding the standardization of the MSCs treatment. A lack of standard culture procedures makes it hard to compare the clinical outcome of the various studies. MSCs are not static and react to environmental clues. They are living production factories on their own that react to environmental clues and in response change their behavior. Bringing these cells in a robust and reproducible production regime together with the development of standardized bioreactors is highly needed. Standardizing the culture method for a particular clinical application could result in less variation in the outcome and boost the clinical application in the future.

Acknowledgements

None.

Conflicts of interest

R.V. is CSO of DeltaLife, a CMO for the production of ATMPs.

There are no conflicts of interest.

REFERENCES AND RECOMMENDED READING

Papers of particular interest, published within the annual period of review, have been highlighted as:

• ▪ of special interest
• ▪▪ of outstanding interest

Additional references related to this topic can also be found in the Current World Literature section in this issue (pp. 116–118).